The electrostatic interaction of cations (Mn 2 ion, Ca 2 ion and cytochrome c) with phospholipid vesicles and other negatively charged membranes will be investigated. The influence of membrane composition, salts, temperature and added perturbants, including anesthetics, will be examined. Through observed changes in cation binding correlated with shifts in lipid fluidity, factors lending thermodynamic stability to membrane structure may be elucidated. MN 2 ion is useful for these studies because of its chemical similarity to Ca 2 ion and its desirable electron paramagnetic resonance (EPR) spectral properties, which not only allow binding to be readily and accurately quantitated over a wide range of conditions, but which can also sometimes yield evidence regarding divalent cation mobility at negatively charged surfaces. This evidence may, in turn, provide a basis for distinguishing spectrally among various classes of membrane binding sites (e.g., phospholipids and sialic acids). The fluidity and permeability of reconstituted glycophorin-phospholipid vesicles, as influenced by added anesthetics, will be measured in order to obtain information on: (1) the molecular mechanism of anesthesia and (2) the nature of lipid-protein interactions in membranes. In this and in other parts of the proposed work, membrane fluidity will be monitored with nitroxide spin labels. The location of glycophorin tyrosines in the vesicles will be probed with fluorescence. A better understanding of protein conformation and orientation in membranes can be gained in this way. Efforts will be made, through fluorescence and EPR, to detect amplified changes in the organization of plasma membranes induced by binding of hormones or other perturbants and subsequent lateral information transfer in the plane of the membrane. Where such changes are observed, attempts will be made to localize the affected components as to depth in the membrane.